Viral vectors

ABSTRACT

The present invention provides a herpes virus which lacks a functional ICP34.5 encoding gene and which comprises two or more of—(i) a gene encoding a prodrug converting enzyme; (ii) a gene encoding a protein capable of causing cell to cell fusion; and (iii) a gene encoding an immunomodulatory protein.

FIELD OF THE INVENTION

The present invention relates to herpes virus strains with improvedanti-tumour activity as compared to previously known strains.

BACKGROUND TO THE INVENTION

Viruses have been shown to have utility in a variety of applications inbiotechnology and medicine on many occasions. Each is due to the uniqueability of viruses to enter cells at high efficiency. This is followedin such applications by either virus gene expression and replicationand/or expression of an inserted heterologous gene. Thus viruses caneither deliver and express viral or other genes in cells which may beuseful in for example gene therapy or the development of vaccines, orthey may be useful in selectively killing cells by lytic replication orthe action of a delivered gene in for example cancer.

Herpes simplex virus (HSV) has been suggested to be of use for theoncolytic treatment of cancer. A virus for use in treating cancer musthowever be disabled such that it is no longer pathogenic, i.e. does notreplicate in and kill non-tumor cells, but such that it can still enterand kill tumor cells. For the oncolytic treatment of cancer, which mayalso include the delivery of gene(s) enhancing the therapeutic effect, anumber of mutations to HSV have been identified which still allow thevirus to replicate in culture or in actively dividing cells in vivo(e.g. in tumors), but which prevent significant replication in normaltissue. Such mutations include disruption of the genes encoding ICP34.5,ICP6 and thymidine kinase. Of these, viruses with mutations to ICP34.5,or ICP34.5 together with mutation of, for example, ICP6, have so farshown the most favourable safety profile. Viruses deleted only for theneurovirulence factor ICP34.5 have been shown to replicate in many tumorcell types in vitro and to selectively replicate in artificially inducedbrain tumors in mice while sparing surrounding tissue. Early stageclinical trials have also shown their safety in man.

SUMMARY OF THE INVENTION

The present invention provides viruses with improved capabilities forthe destruction of tumor cells in vivo. Viruses provided by the presentinvention comprise an inactivating mutation in the gene encoding ICP34.5and are capable of delivering two genes which, in combination, enhancethe therapeutic effect. The virus comprises a gene from two or more ofthe following types:

A gene which encodes a pro-drug activating enzyme capable of convertingan inactive or poorly active prodrug into the active or more activeform. Treatment of tumors with the virus is therefore accompanied byadministration of the prodrug.

A gene which encodes a protein capable of fusing cells (ie causing theformation of syncytia). This itself provides anti-tumour effect, whichmay be mediated by the induction of an immune response. However, incombination with pro-drug activation this anti-tumour effect isenhanced. Such fusogenic genes include modified retroviral envelopeglycoproteins such as those derived from gibbon ape leukaemia virus orhuman endogenous retrovirus W, the fusogenic F and H proteins frommeasles virus or the vesicular stomatitis virus G protein, but othergenes encoding proteins capable of causing cell fusion may also be used.

A gene which encodes an immunomodulatory protein. The immunomodulatoryprotein promotes an anti-tumour immune response. Such gene(s) includeimmune modulators such as GM-CSF, TNFα, CD40L or other cytokines orco-stimulatory molecules. The immunomodulatory protein enhances theeffects of the prodrug activating gene and/or the protein capable ofcausing cell to cell fusion alone. Thus, viruses of the inventioninclude viruses encoding a prodrug activating gene and animmunomodulatory gene, but no protein capable of causing cell to cellfusion, and viruses encoding a protein capable of causing cell to cellfusion and an immunomodulatory gene, but no prodrug converting gene.

The present invention thus provides viruses capable of the oncolyticdestruction of tumor cells in which, when administered to a patient,optionally in combination with a prodrug, the anti-tumor properties ofthe virus are enhanced by the combined actions of the activated prodrugand the fusogenic and/or immunomodulatory protein expressed from theviral genome, or by the combined actions of the fusogenic protein andthe immunomodulatory protein expressed from the viral genome.

Accordingly, the invention provides:

a herpes virus which lacks a functional ICP34.5 encoding gene and whichcomprises two or more of:

(i) a gene encoding a prodrug converting enzyme;

(ii) a gene encoding a protein capable of causing cell to cell fusion;and

(iii) a gene encoding an immunomodulatory protein.

Preferably the herpes virus is one which lacks a functional ICP34.5encoding gene and which comprises:

(i) a gene encoding a prodrug converting enzyme; and

(ii) a gene encoding a protein capable of causing cell to cell fusion.

The invention also provides:

a herpes virus of the invention for use in a method of treatment of thehuman or animal body by therapy.

use of a herpes virus of the invention in the manufacture of amedicament for the treatment of cancer.

a pharmaceutical composition comprising as active ingredient a herpesvirus according to the invention and a pharmaceutically acceptablecarrier or diluent.

a method of treating a tumour in an individual in need thereof byadministering to said individual an effective amount of a herpes virusaccording to the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the plasmids used to construct the viruses of theinvention and illustrates how the plasmids were constrcuted. Theplasmids encode either a cytosine deaminase gene or a gibbon apeleukemia (GALV) fusogenic glycoprotein, or both genes flanked by HSV1sequences flanking the ICP34.5 encoding gene. The plasmids can be usedfor homologous recombination into the HSV1 gene to replace the ICP34.5encoding gene with either the cytosine deaminase gene, the GALVglycoprotein gene or both genes. These plasmids were then used toconstruct the viruses shown in FIG. 2.

FIG. 1 a shows the subcloning of Fcy:Fur gene from pORF Fcy:Fur(Invivogen) to pRcRSV (Invitrogen). Fcy:Fur gene was removed byrestriction digestion (Nhe I and Nco I (blunted with T⁴ polymerase).This fragment was then ligated into pRcRSV (Invitrogen) cut with Xba Iand Hind III (blunted with T⁴ polymerase).

FIG. 1 b shows the subcloning of RSV Fcy:Fur pA cassette from pRcRSVinto p-34.5 by restriction digestion (Pvu II, NruI). This fragment wasthen ligated into the shuttle vector p-34.5 cut with NotI (blunted withT⁴ polymerase). p-34.5 plasmid is based on pSP72 (Promega) and containstwo flanking regions either side of HSV-1 ICP34.5 gene (based onHSV-117+strain (123462-124958 bp, 125713-126790 bp)) Genbank X14112).

FIG. 1 c shows the truncated envelope (env) of Gibbon Ape Leukaemiavirus (Genbank NC_(—)001885, 5552-7555 bp) which was obtained by RT-PCRfrom a viral producer cell line (MLV 144, Rangan et al., 1979). Theenvelope (GALV env R−) was cloned into pcDNA3 (Invitrogen) (byrestriction disgested Eco RI Not I), a mammalian expression vector andthree of the clones were tested for syncytia production.

FIG. 1 d shows the knock out of a Not-1 site in pcDNA3 GALV env R− bydigestion using Not I and blunted with T⁴ polymerase, follow byreligation.

FIG. 1 e shows the subcloning of GALV env R− virus (Genbank NC 001885,5552-7555 bp) from pcDNA3 (Invitrogen) to pGEM T Easy by PCR (Promega).

FIG. 1 f shows the subcloning of GALV env R− from pGEM T Easy (Promega)to p-34.5 by restriction digestion (NotI). p-34.5 plasmid is based onpSP72 (Promega) and contains two flanking regions either side of HSV-1ICP 34.5 gene (based on HSV-117+strain (123462-124958 bp, 125713-126790bp)) Genbank X14112).

FIG. 1 g shows the final shuttle vector containing ICP34.5 flankingregions (based on HSV-117+strain (123462-124958 bp, 125713-126790 bp)Genbank X14112)) and expressing the truncated envelope (env) of GibbonApe Leukaemia virus (Genbank NC_(—)001885, 5552-7555 bp) under a CMVpromoter and BGH poly A (Invitrogen).

FIG. 1 h shows the subcloning of RSV Fcy:Fur pA cassette from pRcRSVinto p-34.5 GALV (**) by restriction digestion (Pvu II, NruI). Thisfragment was then ligated into the shuttle vector p-34.5 GALV cut withNru I. p-34.5 plasmid is based on pSP72 (Promega) and contains twoflanking regions either side of HSV-1 ICP 34.5 gene (based onHSV-117+strain (123462-124958 bp, 125713-126790 bp)) Genbank X14112) andexpressing the truncated envelope (env) of Gibbon Ape Leukaemia virus(Genbank NC_(—)001885, 5552-7555 bp) under a CMV promoter and BGH poly A(Invitrogen).

FIG. 2 is a schematic representation of virus vectors used in thisstudy. HSV-1 strain JS1 was isolated by taking a swab from a cold soreof a otherwise healthy volunteer (Liu et al 2003). JS1/34.5−/47− has twodeletions. The first involves removal of the coding region of theICP34.5 gene (nucleotides 124948-125713 based on the sequence HSV-1strain 17+). The second involves a 280 bp deletion of ICP47 (nucleotides145570-145290 based on the sequence HSV-1 strain 17+) (Liu et al 2003).JS1/34.5−/GALVenv R−/47− expresses the retroviral envelope of gibbon apeleukaemia virus—the R-peptide (Genbank NC_(—)001885, 5552-7555 bp)(Bateman et al 2000, Galanis et al 2001) under CMV promoter.JS1/34.5−/RSV/Fcy:Fur/47− expresses the enzyme prodrug activator yeastcytosine deaminase fusion to uracil phospo-ribosyltransferase(Invivogen) under RSV promoter. JS1/34.5−/GALV/env R−/Fcy:Fur/47−combines both fusogenic retroviral envelope and enzyme prodrugactivator.

FIG. 3 shows the fusion of tumour cells by GALV env R−. A plaque isshown of JS1/34.5−/47− (A) and JS1/34.5−/47−/GALV (B) followinginfection of rat RG2 cells and staining with crystal violet.

FIG. 4 shows the effect of supernatant from HT1080 cells 48 hours afterinfection with the control virus, JS1/34.5−/47−, JS1/34.5−/47−/Fcy:Furor JS1/34.5−/47−/GALV/Fcy:Fur, in the presence or absence of5-fluorocytosine (5-FC). These supernatants were then heat inactivatedand then added to fresh HT1080 cells for 72 hours. The two lower righthand panels show near complete cell death indicating that5-fluorocytosine has been converted to 5-fluorouracil by the Fcy:Furcontaining viruses.

FIG. 5 shows the effect of JS1/34.5−/47−, JS1/34.5−/47−/GALV,JS1/34.5−/47−/Fcy:Fur viruses on shrinking tumours implanted in rats.Rat 9L tumour cells were implanted into the flank of Fischer rats andallowed to develop to give a tumour diameter of approx 4-5 mm. Groups offive rats were then injected with 50 μl of 1×10 esp8 pfu/ml theindicated virus, intratumorally, on days 7, 10, 13, 17 and 20. 500 mg/kgof 5-fluorocytosine was administered by the intraperitoneal route ondays 9, 11, 12, 14, 16, 18, 19, 21, 23, 24, 25, 26 and tumour diametersmeasured. Error bars represent standard deviation.

DETAILED DESCRIPTION OF THE INVENTION

A. Viruses

A herpes virus of the invention is capable of efficiently infectingtarget tumor cells. The genes encoding ICP34.5 are inactivated in thevirus. Mutation of ICP34.5 allows selective oncolytic activity. Suitablemutations in the ICP34.5 genes are described in Chou et al 1990 andMaclean et al 1991, although any mutation which renders ICP34.5 isnon-functional may be used. The genes encoding ICP47, ICP6 and/orthymidine kinase may additionally be inactivated, as may other genes ifsuch inactivation does significantly reduce the oncolytic effect, or ifsuch deletion enhances oncolytic or other desirable properties of thevirus. Where the gene encoding ICP47 is mutated it may be mutated insuch a fashion that the nearby US11 gene is expressed at earlier timesin the HSV replication cycle than is usually the case. Such a mutationis described in Liu et al 2003. Viruses of the invention additionallyencode two or more of a prodrug activating enzyme, a protein capable ofcausing cell to cell fusion and an immunomodulatory protein.

The terminology used herein for the herpes virus genes is that commonlyused for genes of HSV. Where the herpes virus of the invention is from anon-HSV herpes virus the functional equivalent of each of the mentionedHSV genes is inactivated. A non-HSVgene which is a functional equivalentof an HSV gene perfoms the same function as the HSV gene and shares adegree of sequence homology with the HSV gene. The functional equivalentmay be at least 30%, for example at least 40% or at least 50%,homologous to the HSV gene. Homology may be determined as describedbelow.

Viral regions altered for the purposes described above may be eithereliminated (completely or partly), or made non-functional, orsubstituted by other sequences, in particular by a gene encoding aprodrug converting enzyme, a gene encoding a protein capable of causingcell to cell fusion or a gene encoding an immunomodulatory protein

The virus of the invention may be derived from a herpes simplex virusHSV strain. The HSV strain may be an HSV1 or HSV2 strain, or aderivative thereof, and is preferably HSV1. Derivatives includeinter-type recombinants containing DNA from HSV1 and HSV2 strains. Suchinter-type recombinants are described in the art, for example inThompson et al 1998 and Meignier et al 1988. Derivatives preferably haveat least 70% sequence homology to either the HSV1 or HSV2 genome, morepreferably at least 80%, even more preferably at least 90 or 95%. Morepreferably, a derivative has at least 70% sequence identity to eitherthe HSV1 or HSV2 genome, more preferably at least 80% identity, evenmore preferably at least 90%, 95% or 98% identity.

For example the UWGCG Package provides the BESTFIT program which can beused to calculate homology (for example used on its default settings)(Devereux et al. (1984) Nucleic Acids Research 12, p 387-395). ThePILEUP and BLAST algorithms can be used to calculate homology or line upsequences (typically on their default settings), for example asdescribed in Altschul (1993) J. Mol. Evol. 36:290-300; Altschul et al.(1990) J. Mol. Biol. 215:403-10.

Software for performing BLAST analyses is publicly available through theNational Centre for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pair (HSPs) by identifying short wordsof length W in the query sequence that either match or satisfy somepositive-valued threshold score T when aligned with a word of the samelength in a database sequence. T is referred to as the neighbourhoodword score threshold (Altschul et al., 1990). These initialneighbourhood word hits act as seeds for initiating searches to findHSPs containing them. The word hits are extended in both directionsalong each sequence for as far as the cumulative alignment score can beincreased. Extensions for the word hits in each direction are haltedwhen: the cumulative alignment score falls off by the quantity X fromits maximum achieved value; the cumulative score goes to zero or below,due to the accumulation of one or more negative-scoring residuealignments; or the end of either sequence is reached. The BLASTalgorithm parameters W, T and X determine the sensitivity and speed ofthe alignment. The BLAST program uses as defaults a word length (W) of11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc.Natl. Acad. Sci. USA 89: 10915-10919) alignments (B) of 50, expectation(E) of 10, M=5, N=4, and a comparison of both strands.

The BLAST algorithm performs a statistical analysis of the similaritybetween two sequences; see e.g., Karlin and Altschul (1993) Proc. Natl.Acad. Sci. USA 90: 5873-5787. One measure of similarity provided by theBLAST algorithm is the smallest sum probability (P(N)), which providesan indication of the probability by which a match between two nucleotideor amino acid sequences would occur by chance. For example, a sequenceis considered similar to another sequence if the smallest sumprobability in comparison of the first sequence to the second sequenceis less than about 1, preferably less than about 0.1, more preferablyless than about 0.01, and most preferably less than about 0.001.

A derivative may have the sequence of a HSV1 or HSV2 genome modified bynucleotide substitutions, for example from 1, 2 or 3 to 10, 25, 50 or100 substitutions. The HSV1 or HSV2 genome may alternatively oradditionally be modified by one or more insertions and/or deletionsand/or by an extension at either or both ends.

Virus strains of the invention may be “non-laboratory” strains. Thesecan also be referred to as “clinical” strains. A person of skill in theart will readily be able to distinguish between a laboratory strain anda non-laboratory, or clinical, strain. Further guidance on theproperties likely to be exhibited by virus strains is given below.

The key distinction between a laboratory and non-laboratory strain isthat laboratory strains currently in common use have been maintained forlong periods, many years in some cases, in culture. All laboratory HSVstrains will originally have been isolated from infected individuals andso are derived from clinical strains. The culture of viruses such as HSVinvolves a technique known as serial passage. To grow and maintainviruses, suitable cells are infected with the virus, the virusreplicates within the cell and the virus is then harvested; fresh cellsare then re-infected, this process constitutes one cycle of serialpassage. Each such cycle may take, for example, a few days in the caseof HSV. As discussed above, such serial passaging may lead to changes inthe properties of the virus strain, in that selection takes places forproperties that favour growth in culture (e.g. rapid replication), asopposed to properties useful for practical applications, e.g.maintenance of the capacity to travel along axons in the case of HSV orto infect human cells.

Laboratory strains in current use include HSV-1 strain F, HSV-1 strain17+ and HSV-1 strain KOS. Non-laboratory strains useful in the inventiontypically have improved oncolytic activity compared to HSV-1 strains F,17+ and KOS strains with equivalent modifications.

A non-laboratory strain is one that has been recently isolated from aninfected individual. A non-laboratory strain of the present invention isa recently isolated strain that has been modified so that the geneencoding ICP34.5 is inactivated such that a functional ICP34.5 proteincannot be expressed and to include a gene encoding a pro-drug activatingprotein, a gene encoding a protein capable of causing cell fusion and/oran immunomodulatory protein. A virus of the invention will have spentsome time in culture in order to allow the necessary modifications to bemade, but any time spent in culture will be comparatively short. Theclinical isolate may have been frozen for storage prior to modification,or may be frozen after modifications have been made. Strains of theinvention are prepared in such a manner so as to retain substantiallythe desirable properties of the original clinical isolates from whichthey are derived.

A virus strain of the invention is derived from a parental virus strainif the parental virus strain is mutated to produce the virus. Forexample, a virus of the invention may be derived from the clinicalisolate JS1. The parental strain of such a JS1-derived virus may be JS1or another HSV1 strain derived from JS1. Thus a virus of the inventionmay be a JS1 virus which lacks a functional ICP34.5 encoding gene andwhich comprises two or more of a gene encoding a prodrug convertingenzyme, a gene encoding a protein capable of causing cell to cell fusionand a gene encoding an immunomodulatory protein. In addition, such avirus may contain any other mutation, for example, as mentioned herein.

A virus of the invention is capable of efficiently infecting targethuman cancer cells. When such a virus is a non-laboratory or clinicalstrain it will have been recently isolated from an HSV infectedindividual and then screened for the desired ability of enhancedreplication, infection or killing of tumour and/or other cells in vitroand/or in vivo in comparison to standard laboratory strains such asHSV-1 strains F, KOS and 17+. Such viruses of the invention withimproved properties as compared to laboratory virus strains are thenengineered such that they lack functional a ICP34.5 gene and encode twoor more of the following genes: a gene for a prodrug activating enzyme,a gene for a protein capable of causing cell to cell fusion and a geneencoding an immunomodulatory protein wherein said genes are under thecontrol of a suitable promoter(s). A virus strain has been recentlyisolated if it has undergone three years or less in culture sinceisolation of the unmodified clinical parent strain from its host. Morepreferably, the strain has undergone one year or less in culture, forexample nine months or less, six months or less, three months or less,two months or less, one month or less, two weeks or less, or one week orless. By these definitions of time in culture, is meant time actuallyspent in culture. Thus, for example, it is a common practice to freezevirus strains in order to preserve them. Evidently, preserving byfreezing or in an equivalent manner does not qualify as maintaining thestrain in culture. Thus, time spent frozen or otherwise preserved is notincluded in the above definitions of time spent in culture. Time spentin culture is typically time actually spent undergoing serial passage,i.e. time during which selection for undesirable characteristics canoccur.

Preferably, a non-laboratory virus strain has undergone 1,000 or lesscycles or serial passage since isolation of its unmodified clinicalprecursor strain from its host. More preferably, it has undergone 500 orless, 100 or less, 90 or less, 80 or less, 70 or less, 60 or less, 50 orless, 40 or less, 30 or less, 20 or less, 10 or less, 5 or less, 4 orless, 3 or less, 2 or 1 such cycles.

Preferably, a non-laboratory virus has a greater ability, as measured bystandard statistical tests, than a reference laboratory strain with theequivalent modifications to perform certain functions useful in theapplication at hand. In the case of an oncolytic virus for tumourtreatment, a non-laboratory virus strain of the invention willpreferably have a greater ability than a reference laboratory strainwith equivalent modifications to infect or replicate in tumour cells, tokill tumour cells or to spread between cells in tissue. More preferably,such greater ability is a statistically significantly greater ability.For example, according to the invention, a non-laboratory strain of theinvention may have up to 1.1 fold, 1.2 fold, 1.5 fold, 2 fold, 5 fold,10 fold, 20 fold, 50 fold, or 100 fold the capacity of the referencestrain in respect of the property being tested. Preferably, thereference strain is selected from HSV-1 strain 17+, HSV-1 (F) and HSV-1KOS.

Statistical analysis of the properties described herein may be carriedout by standard tests, for example, t-tests, ANOVA, or Chi squaredtests. Typically, statistical significance will be measured to a levelof p=0.05 (5%), more preferably p=0.01p, p=0.001, p=0.0001, p=0.000001.

Viruses of the invention infect and replicate in tumour cells,subsequently killing the tumour cells. Thus, such viruses arereplication competent. Preferably, they are selectively replicationcompetent in tumour cells. This means that either they replicate intumour cells and not in non-tumour cells, or that they replicate moreeffectively in tumour cells than in non-tumour cells. For example, wherethe virus is used for treating a tumor in the central nervous system,the virus is capable of replicating in the tumor cells but not in thesurrounding neuronal cells. Cells in which the virus is able toreplicate are permissive cells. Measurement of selective replicationcompetence can be carried out by the tests described herein formeasurement of replication and tumour cell-killing capacity, and alsoanalysed by the statistical techniques mentioned herein if desired.

The properties of the virus strain in respect of tumour cells can bemeasured in any manner known in the art. For example, the capacity of avirus to infect a tumour cell can be quantified by measuring the dose ofvirus required to measure a given percentage of cells, for example 50%or 80% of cells. The capacity to replicate in a tumour cell can bemeasured by growth measurements such as those carried out in theExamples, e.g. by measuring virus growth in cells over a period of 6,12, 24, 36, 48 or 72 hours or longer.

The ability of a virus to kill tumour cells can be roughly quantitatedby eye or more exactly quantitated by counting the number of live cellsthat remain over time for a given time point and MOI for given celltype. For example, comparisons may be made over 24, 48 or 72 hours andusing any known tumour cell type. In particular, HT29 colorectaladenocarcinoma, LNCaP.FGC prostate adenocarcinoma, MDA-MB-231 breastadenocarcinoma, SK-MEL-28 malignant melanoma or U-87 MG glioblastomaastrocytoma cells can be used. Any one of these cell types or anycombination of these cell types can be used, as may other tumour celltypes. It may be desirable to construct a standard panel of tumour celltypes for this purpose. To count the number of live cells remaining at agiven time point, the number of trypan blue-excluding cells (i.e. livecells) can be counted. Quantitation may also be carried out byfluorescence activated cell sorting (FACS) or MTT assay. Tumourcell-killing ability may also be measured in vivo, e.g. by measuring thereduction in tumour volume engendered by a particular virus.

In order to determine the properties of viruses of the invention, itwill generally be desirable to use a standard laboratory referencestrain for comparison. Any suitable standard laboratory reference strainmay be used. In the case of HSV, it is preferred to use one or more ofHSV1 strain 17+, HSV1 strain F or HSV1 strain KOS. The reference strainwill typically have equivalent modifications to the strain of theinvention being tested. Thus, the reference strain will typically haveequivalent modifications, such as gene deletions and heterologous geneinsertions. In the case of a virus of the invention, where the ICP34.5encoding genes have been rendered non-functional, the ICP34.5 encodinggenes will also have been rendered non-functional in the referencestrain. The modifications made to the reference strain may be identicalto those made to the strain of the invention. By this, it is meant thatthe gene disruptions in the reference strain will be in exactlyequivalent positions to those in the strain of the invention, e.g.deletions will be of the same size and in the same place. Similarly, inthese embodiments, heterologous genes will be inserted in the sameplace, driven by the same promoter, etc. However, it is not essentialthat identical modifications be made. What is important is that thereference gene has functionally equivalent modifications, e.g. that thesame genes are rendered non-functional and/or the same heterologous geneor genes is inserted.

B. Methods of Mutation

The various genes referred to may be rendered functionally inactive byseveral techniques well known in the art. For example, they may berendered functionally inactive by deletion(s), substitution(s) orinsertion(s), preferably by deletion. Deletions may remove one or moreportions of the gene or the entire gene. For example, deletion of onlyone nucleotide may be made, resulting in a frame shift. However,preferably a larger deletion(s) is made, for example at least 25%, morepreferably at least 50% of the total coding and non-coding sequence (oralternatively, in absolute terms, at least 10 nucleotides, morepreferably at least 100 nucleotides, most preferably, at least 1000nucleotides). It is particularly preferred to remove the entire gene andsome of the flanking sequences. Where two or more copies of the gene arepresent in the viral genome it is preferred that both copies of the geneare rendered functionally inactive.

Mutations are made in the herpes viruses by homologous recombinationmethods well known to those skilled in the art. For example, HSV genomicDNA is transfected together with a vector, preferably a plasmid vector,comprising the mutated sequence flanked by homologous HSV sequences. Themutated sequence may comprise a deletion(s), insertion(s) orsubstitution(s), all of which may be constructed by routine techniques.Insertions may include selectable marker genes, for example lacZ orgreen fluorescent protein (GFP), which may be used for screeningrecombinant viruses, for example, β-galactosidase activity orfluorescence.

C. Heterologous Genes and Promoters

The viruses of the invention carry a two or more of a heterologous geneencoding a prodrug activating enzyme, a heterologous gene encoding aprotein capable of causing cell to cell fusion and a heterologous geneencoding an immunomodulatory protein. Preferably a virus of theinvention comprises a heterologous gene encoding a prodrug activatingenzyme and one or both of a heterologous gene encoding a fusogenicprotein and a heterologous gene encoding an immunomodulatory protein.The fusogenic protein may also function as an immunomodulatory protein.

Preferably, the prodrug activating protein is a cytosine deaminaseenzyme. Cytosine deaminase genes are capable of converting the inactiveprodrug 5-fluorocytosine to the active drug 5-flurouracil. Variouscytosine deaminase genes are available including those of bacterialorigin and of yeast origin. A second gene, typically a gene encoding asecond enzyme, may be used to enhance the prodrug conversion activity ofthe cytosine deaminase gene. For example, the second gene may encode auracil phosphoribosyltransferase like the viruses described in FIG. 2.

Any suitable fusogenic gene encoding a protein capable of causing cellcell fusion may be used. Preferably the protein capable of causing cellto cell fusion is selected from a modified retroviral envelopeglycoprotein, such as an envelope glycoprotein derived from gibbon apeleukaemia virus (GALV) or human endogenous retrovirus W, a fusogenic For H protein from measles virus and the vesicular stomatitis virus Gprotein. More preferably, the protein capable of causing cell to cellfusion is a GALV fusogenic glycoprotein.

The immunomodulatory gene may be any gene encoding a protein that iscapable of modulating an immune response. The protein capable ofmodulating an immune response may be a cytokine, such as GM-CSF, TNF-α,an interleukin (for example IL12), a chemokine such as RANTES or amacrophage inflammatory protein (for example MIP-3) or anotherimmunomodulatory molecule such as B7.1, B7.2 or CD40L The proteincapable of causing cell to cell fusion may also be capable of modulatingan immune response. For example, GALV is capable of modulating an immuneresponse.

Viruses of the invention may thus be used to deliver the genes to a cellin vivo where they will be expressed.

The prodrug activating gene, the gene encoding a protein capable ofcausing cell to cell fusion and/or the gene encoding an immunomodulatoryprotein may be inserted into the viral genome by any suitable techniquesuch as homologous recombination of HSV strains with, for example,plasmid vectors carrying the gene flanked by HSV sequences. The genesmay be inserted at the same site in the HSV genome, for example so as toreplace the ICP34.5 encoding gene, or at different sites. The genes maybe expressed from separate promoters, for example a CMV promoter and anRSV promoter or from a single promoter. Where the genes are expressedfrom a single promoter, the genes may be separated by an internalribosome entry site (IRES). The genes may also be expressed as atranslational fusion such that the fused protein retains both activitiesof the separate genes (ie prodrug activation and cell to cell fusion,prodrug activation and immunomodulatory activity or cell to cell fusionand immunomodulatory activity) such that the fused proteins are cleavedfollowing expression by a protease either in cis or in trans to thefused protein. In a preferred embodiment, the two proteins, or two ofthe three proteins, are expressed from an RSV and a CMV promoterrespectively placed in a back-to-back orientation with respect to eachother and inserted into the HSV genome so as to replace the genesencoding ICP34.5. Such a virus is described in FIG. 2. However, the genemay be inserted into the viral genome at any location(s) provided thatoncolytic properties are retained.

The transcribed sequences of the inserted genes are preferably operablylinked to control sequences permitting expression of the genes in atumour cell. The term “operably linked” refers to a juxtapositionwherein the components described are in a relationship permitting themto function in their intended manner. A control sequence “operablylinked” to a coding sequence is ligated in such a way that expression ofthe coding sequence is achieved under conditions compatible with thecontrol sequence.

A control sequence typically comprises a promoter allowing expression ofthe gene operably linked thereto and signal for termination oftranscription. The promoter is selected from promoters which arefunctional in mammalian, preferably human tumour cells. The promoter maybe derived from promoter sequences of a eukaryotic gene. For example,the promoter may be derived from the genome of a cell in whichexpression of the heterologous gene is to occur, preferably a mammaliantumour cell, more preferably a human tumour cell. With respect toeukaryotic promoters, they may be promoters that function in aubiquitous manner (such as promoters of β-actin, tubulin) or,alternatively, in a tumour-specific manner. They may also be promotersthat respond to specific stimuli, for example promoters that bindsteroid hormone receptors. Viral promoters may also be used, for examplethe Moloney murine leukaemia virus long terminal repeat (MMLV LTR)promoter or other retroviral promoters such as that derived from Roussarcoma virus (RSV), the human or mouse cytomegalovirus (CMV) IEpromoter or promoters of herpes virus genes including those drivingexpression of the latency associated transcripts.

Expression cassettes and other suitable constructs comprising theprodrug converting enzyme encoding gene, gene encoding a protein capableof promoting cell to cell fusion and/or immunomodulatory gene andcontrol sequences can be made using routine cloning techniques known topersons skilled in the art (see, for example, Sambrook et al., 1989,Molecular Cloning—A laboratory manual; Cold Spring Harbor Press).

It may also be advantageous for the promoter(s) to be inducible so thatthe levels of expression of the genes can be regulated during thelife-time of the tumour cell. Inducible means that the levels ofexpression obtained using the promoter can be regulated. For example, avirus of the invention may further comprise a heterologous gene encodingthe tet repressor/VP16 transcriptional activator fusion protein underthe control of a strong promoter (e.g. the CMV IE promoter) and theprodrug converting, cell to cell fusion or immunomodulatory or othergene may be under the control of a promoter responsive to the tetrepressor VP16 transcriptional activator fusion protein previouslyreported (Gossen and Bujard, 1992, Gossen et al, 1995). Thus, in thisexample, expression of the gene(s) would depend on the presence orabsence of tetracycline.

Viruses of the invention encode multiple heterologous genes. Viruses ofthe invention may comprise one or more additional genes, for examplefrom 1, 2 to 3, 4 or 5 additional genes. The additional gene(s) may befurther copies of the prodrug converting gene, the fusiogenic geneand/or the immunomodulatory gene. The additional gene(s) may encodes oneor more different prodrug converting gene, one or more differentfusiogenic gene and/or one or more different immunomodulatory gene. Theadditional gene(s) may encodes other gene(s) intended to enhance thetherapeutic effect.

More than one gene and associated control sequences could be introducedinto a particular HSV strain either at a single site or at multiplesites in the virus genome. Alternatively pairs of promoters (the same ordifferent promoters) facing in opposite orientations away from eachother, each driving the expression of a gene may be used.

D. Therapeutic Uses

Viruses of the invention may be used in a method of treating the humanor animal body. In particular, viruses of the invention may be used inmethods of cancer therapy. Preferably, viruses of the invention are usedin the oncolytic treatment of cancer. Viruses of the invention may beused in the therapeutic treatment of any solid tumour in a mammal,preferably a human. For example viruses of the invention may beadministered to a subject with prostate, breast, lung, liver, renalcell, endometrial, bladder, colon or cervical carcinoma; adenocarcinoma;melanoma; lymphoma; glioma; sarcomas such as soft tissue and bonesarcomas; or cancer of the head and neck.

E. Administration

The viruses of the invention may be used in a patient, preferably ahuman patient, in need of treatment. A patient in need of treatment isan individual suffering from cancer, preferably an individual with asolid tumour. The aim of therapeutic treatment is to improve thecondition of a patient. Typically therapeutic treatment using a virus ofthe invention allieviates the symptoms of the cancer. A method oftreatment of cancer according to the invention comprises administering atherapeutically effective amount of a virus of the invention to apatient suffering from cancer. Administration of an oncolytic virus ofthe invention to an individual suffering from a tumour will typicallykill the cells of the tumour thus decreasing the size of the tumourand/or preventing spread of malignant cells from the tumour.

One method of administering therapy involves combining the virus with apharmaceutically acceptable carrier or diluent to produce apharmaceutical composition. Suitable carriers and diluents includeisotonic saline solutions, for example phosphate-buffered saline.

Therapeutic treatment may be carried out following direct injection ofthe virus composition into target tissue. The target tissue may be thetumour or a blood vessel supplying the tumour. The amount of virusadministered is in the case of HSV in the range of from 10⁴ to 10¹⁰ pfu,preferably from 10⁵ to 10⁸ pfu, more preferably about 10⁶ to 10⁹ pfu.Typically 1-4 ml, such as 2 to 3 ml of a pharmaceutical compositionconsisting essentially of the virus and a pharmaceutically acceptablesuitable carrier or diluent would be used for direct injection into anindividual tumour. However for some oncolytic therapy applicationslarger volumes up to 10 ml may also be used, depending on the tumourtype, tumour size and the inoculation site. Likewise, smaller volumes ofless than 1 ml may also be used.

The routes of administration and dosages described are intended only asa guide since a skilled practitioner will be able to determine readilythe optimum route of administration and dosage. The dosage may bedetermined according to various parameters, especially according to thelocation of the tumour, the size of the tumour, the age, weight andcondition of the patient to be treated and the route of administration.Preferably the virus is administered by direct injection into thetumour. The virus may also be administered systemically or by injectioninto a blood vessel supplying the tumour. The optimum route ofadministration will depend on the location and size of the tumour.

THE FOLLOWING EXAMPLES ILLUSTRATE THE INVENTION

In work aimed at producing ICP34.5 deleted HSV with enhanced oncolyticand anti-tumour potential, we have deleted ICP47 and ICP34.5 from HSV1strain JS1 and have inserted the genes encoding a prodrug activatinggene (a cytosine deaminase/uracil phosphoribosyltransferase fusion gene)and/or a gene for a GALV fusogenic glycoprotein.

Example 1 Virus Construction (see FIGS. 1 & 2)

The viruses used were based on the clinical, or “non-laboratory”, HSV1strain, JS1. ICP34.5 and ICP47 were completely deleted from strain JS1.This virus is described in Lui et al 2003. The GALV env R− (Bateman etal 2000, Galanis et al 2001) and/or the cytosine deaminase/uracilphosphoribosyltransferase fusion gene (Fcy:Fur; Invitrogen) were theninserted in place of the ICP34.5 encoding gene under CMV and RSVpromoter control respectively.

FIGS. 1 a-1 h demonstrate the stepwise construction of the plasmids usedto construct the viruses:

Step 1 (FIG. 1 a): The Fcy::Fur gene was excised from pORF Fcy::Fur withNcoI and NheI and inserted into pRCRSV following digestion with HindIIIand XbaI;

Step 2 (FIG. 1 b): The RSV promoter/Fcy::Fur/BGHpA cassette from PRcRSVFcy::Fur was inserted between ICP34.5 flanking regions (Lui et al 2003)to generate p-34.5 Fcy::Fur;

Step 3 (FIG. 1 c): The GALV env R− was amplified by PCR from cellscontaining the integrated provirus and cloned into pcDNA3 between theNotI and EcoRI sites to generate pcDNA3 kGALV env R−;

Step 4 (FIG. 1 d-1 e): Manipulation to remove restriction sites whichwere not required;

Step 4 (FIG. 1 g): The CMV promoter/GALV R−/BGHpA cassette was clonedinto ICP34.5 flanking regions to generate p-34.5 CMV GALV env R−;

Step 5 (FIG. 1 h): To generate a plasmid allowing insertion in place ofICP34.5 and containing both GALV env R− and Fcy::Fur genes, the RSVpromoter/Fcy::Fur/pA cassette from pRcRSV Fcy::Fur was excised andinserted into p-34.5 CMV GALV env R− to generate p-34.5 GALV FCY.

Plasmids p-34.5 Fcy::Fur, p-34.5 CMV GALV env R− and p-34.5 GALV FCYwere inserted into virus strain JS1/34.5−/47− CMV GFP (Lui et al 2003)by homologous recombination so as to replace the GFP sequence replacingICP34.5. Recombinant, non-GFP expressing plaques were selectedgenerating three viruses (JS1/34.5−/47−/Fcy::Fur, JS1/34.5−/47−/GALV andJS1/34.5−/47−/Fcy::Fur). These are shown in FIG. 2.

Example 2 The GALV env R− Expressing Viruses Mediate Cell to Cell Fusion

The GALV alone expressing virus (i) causes cell to cell fusion of anumber of human tumour cell lines in vitro, including HT1080, Fadu andU87MG, mediated by the expression of the GALV protein, and (ii) providesincreased anti-tumour activity in vivo in mouse models (Fadu and HT1080)as compared to the equivalent virus not expressing the GALV protein.

Cell to cell fusion is demonstrated in FIG. 3. Rat RG2 glioma cells wereinfected either with JS1/34.5−/47− or JS1/34.5−/47−/GALV and effects onplaque morphology observed. It can be seen that the GALV expressingvirus produces greatly enlarged plaques with signs of a syncitial (cellto cell fusion) effect easily being observed.

Example 3 The Fcy::Fur Expressing Viruses Demonstrate Cytosine DeaminaseActivity

The cytosine deaminase/uracil phosphoribosyltransferase fusion genecontaining virus has demonstrated that it directs the conversion of5-fluorocytosine to 5-fluorouracil in vitro such that 5-fluoruracilmediated cell killing occurs. This is shown in FIG. 4 where HT1080 cellswere infected with the three viruses in the presence or absence of5-fluorocytosine. Supernatants from these cells were then heat treatedto inactivate the virus present in the supernatants. These supernatantswere then used to overlay new cells. If 5-fluorocytosine had beenconverted to the toxic 5-fluorouracil these new cells would then bekilled. It can be seen from the FIG. 4 that when the virus used toinfect the original HT1080 cells contained the Fcy::Fur gene, the cellsonto which the resulting supernatants were overlaid were killeddemonstrating the biological activity of the Fcy::Fur gene.

Example 4 The Combination of GALV env R− and Fcy::Fur ExpressionCombined with 5-fluorocytosine Expression Provides Enhanced Anti-TumourActivity In Vivo as Compared to the Use of Either Gene Alone

FIG. 5 shows the effects of three viruses (JS1/34.5−/47−/GALV,JS1/34.5/47−/Fcy::Fur and JS1/34.5−/47−/GALV/Fcy::Fur) and an ‘emptyvector’ control (JS1/34.5−/47−) on shrinking tumours implanted in theflanks of rats. The viruses were administered in combination with5-fluorocytosine. It can be seen from FIG. 5 that each of the virusescauses shrinkage of the injected tumours. However, while delivery ofeither GALV env R− or Fcy::Fur alone gives improved tumour shrinkage ascompared to the empty vector control, the combined delivery of both GALVenv R− and Fcy::Fur gives still further improved tumour shrinkageeffects, with all tumours in this case being cured. It can be concluded,therefore, that co-delivery of a pro-drug activating gene and afusogenic glycoprotein gives improvements with respect to turnourtherapy as compared to either of the approaches when used alone.

Deposit Information

HSV1 strain JS1 has been deposited at the European Collection of CellCultures (ECACC), CAMR, Sailsbury, Wiltshire SP4 0JG, United Kingdom, on2 Jan. 2001 under provisional accession number 01010209.

REFERENCES

-   Chou et al. 1990, Science 250: 1262-1266-   Maclean et al. 1991, J. Gen. Virol. 72: 631-639-   Gossen M & Bujard H, 1992, PNAS 89: 5547-5551-   Gossen M et al. 1995, Science 268: 1766-1769-   Thompson et al. 1998, Virus Genes 1(3); 275-286-   Meignier et al. 1988, Infect. Dis. 159; 602-614-   Liu et al et al 2003, Gene Therapy 10; 292-303-   Bateman et al 2000, Cancer Research 60; 1492-1497-   Galanis et al 2001, Human Gene Therapy 12; 811-821

1. A herpes virus which lacks a functional ICP34.5 encoding gene andwhich comprises two or more of: (i) a heterologous gene encoding aprodrug converting enzyme; (ii) a heterologous gene encoding a proteincapable of causing cell to cell fusion; and (iii) a heterologous geneencoding an immunomodulatory protein.
 2. A herpes virus according toclaim 1, which comprises: (i) a heterologous gene encoding a prodrugconverting enzyme; and (ii) a heterologous gene encoding a proteincapable of causing cell to cell fusion.
 3. A virus according to claim 1wherein said prodrug converting enzyme is a cytosine deaminase.
 4. Avirus according to claim 1 wherein said protein capable of causing cellto cell fusion is a gibbon ape leukaemia fusogenic glycoprotein.
 5. Aherpes virus according to claim 1, which comprises: (i) a heterologousgene encoding a encoding an immunomodulatory protein; and (ii) aheterologous gene encoding a protein capable of causing cell to cellfusion.
 6. A herpes virus according to claim 1, which comprises: (i) aheterologous gene encoding a prodrug converting enzyme; and (ii) aheterologous gene encoding an immunomodulatory protein.
 7. A virusaccording to claim 1 wherein the immunomodulatory protein is GM-CSF,TNFα or CD40L.
 8. A virus according to an) one of the preceding claimsclaim 1 which comprises one or more further heterologous genes capableof enhancing the anti-tumour therapeutic effect of the virus.
 9. A virusaccording to claim 1 which further lacks a functional gene encodingICP47.
 10. A virus according to claim 1 which further lacks a functionalgene encoding ICP6, glycoprotein H and/or thymidine kinase.
 11. A virusaccording to claim 1 which further lacks a gene encoding a functionalprotein capable of inhibiting dendritic cell function.
 12. A virusaccording to claim 11 in which said gene encoding a functional proteincapable of inhibiting dendritic cell function is UL43 or vhs.
 13. Avirus according to claim 1 which is a strain of herpes simplex virus 1or
 2. 14. A virus according to an) one of the preceding claims claim 1which is a non-laboratory virus strain.
 15. A virus according to claim14 which is derived from HSV1 JS1 as deposited at the Europeancollection of cell cultures (ECAAC) under provisional accession number01010209.
 16. (canceled)
 17. (canceled)
 18. (canceled)
 19. Apharmaceutical composition comprising as active ingredient a virusaccording to claim 1 and a pharmaceutically acceptable carrier ordiluent.
 20. A method of treating a tumour in an individual in needthereof by administering to said individual an effective amount of avirus according to claim
 1. 21. A method according to claim 20 whereinsaid virus is administered by direct intra-tumoral inoculation.